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To elucidate new functions of sphingosine (Sph), we demonstrate that the spontaneous elevation of intracellular Sph levels via caged Sph leads to a significant and transient calcium release from acidic stores that is independent of sphingosine 1-phosphate, extracellular and ER calcium levels. This photo-induced Sph-driven calcium release requires the two-pore channel 1 (TPC1) residing on endosomes and lysosomes. Further, uncaging of Sph leads to the translocation of the autophagy-relevant transcription factor EB (TFEB) to the nucleus specifically after lysosomal calcium release. We confirm that Sph accumulates in late endosomes and lysosomes of cells derived from Niemann-Pick disease type C (NPC) patients and demonstrate a greatly reduced calcium release upon Sph uncaging. We conclude that sphingosine is a positive regulator of calcium release from acidic stores and that understanding the interplay between Sph homeostasis, calcium signaling and autophagy will be crucial in developing new therapies for lipid storage disorders such as NPC.
We have previously reported that the mitochondria inhibitor 3-nitropropionic acid (3-NP), induces the expression of DNA damage-regulated autophagy modulator1 (DRAM1) and activation of autophagy in rat striatum. Although the role of DRAM1 in autophagy has been previously characterized, the detailed mechanism by which DRAM1 regulates autophagy activity has not been fully understood. The present study investigated the role of DRAM1 in regulating autophagy flux. In A549 cells expressing wilt-type TP53, 3-NP increased the protein levels of DRAM1 and LC3-II, whereas decreased the levels of SQSTM1 (sequestosome 1). The increase in LC3-II and decrease in SQSTM1 were blocked by the autophagy inhibitor 3-methyl-adenine. Lack of TP53 or knock-down of TP53 in cells impaired the induction of DRAM1. Knock-down of DRAM1 with siRNA significantly reduced 3-NP-induced upregulation of LC3-II and downregulation of SQSTM1, indicating DRAM1 contributes to autophagy activation. Knock-down of DRAM1 robustly decreased rate of disappearance of induced autophagosomes, increased RFP-LC3 fluorescence dots and decreased the decline of LC3-II after withdraw of rapamycin, indicating DRAM1 promotes autophagy flux. DRAM1 siRNA inhibited lysosomal V-ATPase and acidification of lysosomes. As a result, DRAM1 siRNA reduced activation of lysosomal cathepsin D. Similar to DRAM1 siRNA, lysosomal inhibitors E64d and chloroquine also inhibited clearance of autophagosomes and activation of lysosomal cathapsin D after 3-NP treatment. These data suggest that DRAM1 plays important roles in autophagy activation induced by mitochondria dysfunction. DRAM1 affects autophagy through argument of lysosomal acidification, fusion of lysosomes with autophagosomes and clearance of autophagosomes.
Acidification is required for the function of many intracellular organelles, but methods to acutely manipulate their intraluminal pH have not been available. Here we present a targeting strategy to selectively express the light-driven proton pump Arch3 on synaptic vesicles. Our new tool, pHoenix, can functionally replace endogenous proton pumps, enabling optogenetic control of vesicular acidification and neurotransmitter accumulation. Under physiological conditions, glutamatergic vesicles are nearly full, as additional vesicle acidification with pHoenix only slightly increased the quantal size. By contrast, we found that incompletely filled vesicles exhibited a lower release probability than full vesicles, suggesting preferential exocytosis of vesicles with high transmitter content. Our subcellular targeting approach can be transferred to other organelles, as demonstrated for a pHoenix variant that allows light-activated acidification of lysosomes.
Neurodegeneration in Parkinson's disease (PD) can be recapitulated in animals by administration of α-synuclein preformed fibrils (PFFs) into the brain. However, the mechanism by which these PFFs induce toxicity is unknown. Iron is implicated in PD pathophysiology, so we investigated whether α-synuclein PFFs induce ferroptosis, an iron-dependent cell death pathway. A range of ferroptosis inhibitors were added to a striatal neuron-derived cell line (STHdhQ7/7 cells), a dopaminergic neuron-derived cell line (SN4741 cells), and WT primary cortical neurons, all of which had been intoxicated with α-synuclein PFFs. Viability was not recovered by these inhibitors except for liproxstatin-1, a best-in-class ferroptosis inhibitor, when used at high doses. High-dose liproxstatin-1 visibly enlarged the area of a cell that contained acidic vesicles and elevated the expression of several proteins associated with the autophagy-lysosomal pathway similarly to the known lysosomal inhibitors, chloroquine and bafilomycin A1. Consistent with high-dose liproxstatin-1 protecting via a lysosomal mechanism, we further de-monstrated that loss of viability induced by α-synuclein PFFs was attenuated by chloroquine and bafilomycin A1 as well as the lysosomal cysteine protease inhibitors, leupeptin, E-64D, and Ca-074-Me, but not other autophagy or lysosomal enzyme inhibitors. We confirmed using immunofluorescence microscopy that heparin prevented uptake of α-synuclein PFFs into cells but that chloroquine did not stop α-synuclein uptake into lysosomes despite impairing lysosomal function and inhibiting α-synuclein toxicity. Together, these data suggested that α-synuclein PFFs are toxic in functional lysosomes in vitro. Therapeutic strategies that prevent α-synuclein fibril uptake into lysosomes may be of benefit in PD.
Cells acquire essential nutrients from the environment and utilize adaptive mechanisms to survive when nutrients are scarce. How nutrients are trafficked and compartmentalized within cells and whether they are stored in response to stress remain poorly understood. Here, we investigate amino acid trafficking and uncover evidence for the lysosomal transit of numerous essential amino acids. We find that starvation induces the lysosomal retention of leucine in a manner requiring RAG-GTPases and the lysosomal protein complex Ragulator, but that this process occurs independently of mechanistic target of rapamycin complex 1 activity. We further find that stored leucine is utilized in protein synthesis and that inhibition of protein synthesis releases lysosomal stores. These findings identify a regulated starvation response that involves the lysosomal storage of leucine.
Ethambutol is a common medicine used for the treatment of tuberculosis, which can have serious side effects, such as retinal and liver dysfunction. Although ethambutol has been reported to impair autophagic flux in rat retinal cells, the precise molecular mechanism remains unclear. Using various mammalian cell lines, we showed that ethambutol accumulated in autophagosomes and vacuolated lysosomes, with marked Zn(2+) accumulation. The enlarged lysosomes were neutralized and were infiltrated with Zn(2+) accumulations in the lysosomes, with simultaneous loss of acidification. These results suggest that EB neutralizes lysosomes leading to insufficient autophagy, implying that some of the adverse effects associated with EB in various organs may be of this mechanism.
In dendritic cells, the NADPH oxidase 2 complex (NOX2) is recruited to the phagosomal membrane during antigen uptake. NOX2 produces reactive oxygen species (ROS) in the lumen of the phagosome that kill ingested pathogens, delay antigen breakdown and alter the peptide repertoire for presentation to T cells. How the integral membrane component of NOX2, cytochrome b558 (which comprises CYBB and CYBA), traffics to phagosomes is incompletely understood. In this study, we show in dendritic cells derived from human blood-isolated monocytes that cytochrome b558 is initially recruited to the phagosome from the plasma membrane during phagosome formation. Cytochrome b558 also traffics from a lysosomal pool to phagosomes and this is required to replenish oxidatively damaged NOX2. We identified syntaxin-7, SNAP23 and VAMP8 as the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins mediating this process. Our data describe a key mechanism of how dendritic cells sustain ROS production after antigen uptake that is required to initiate T cell responses.
Lysosomes contain various hydrolases that can degrade proteins, lipids, nucleic acids and carbohydrates. We recently discovered "RNautophagy," an autophagic pathway in which RNA is directly taken up by lysosomes and degraded. A lysosomal membrane protein, LAMP2C, a splice variant of LAMP2, binds to RNA and acts as a receptor for this pathway. In the present study, we show that DNA is also directly taken up by lysosomes and degraded. Like RNautophagy, this autophagic pathway, which we term "DNautophagy," is dependent on ATP. The cytosolic sequence of LAMP2C also directly interacts with DNA, and LAMP2C functions as a receptor for DNautophagy, in addition to RNautophagy. Similarly to RNA, DNA binds to the cytosolic sequences of fly and nematode LAMP orthologs. Together with the findings of our previous study, our present findings suggest that RNautophagy and DNautophagy are evolutionarily conserved systems in Metazoa.
Genetic variation around the LRRK2 gene affects risk of both familial and sporadic Parkinson's disease (PD). However, the biological functions of LRRK2 remain incompletely understood. Here, we report that LRRK2 is recruited to lysosomes after exposure of cells to the lysosome membrane-rupturing agent LLOME. Using an unbiased proteomic screen, we identified the motor adaptor protein JIP4 as an LRRK2 partner at the lysosomal membrane. LRRK2 can recruit JIP4 to lysosomes in a kinase-dependent manner via the phosphorylation of RAB35 and RAB10. Using super-resolution live-cell imaging microscopy and FIB-SEM, we demonstrate that JIP4 promotes the formation of LAMP1-negative tubules that release membranous content from lysosomes. Thus, we describe a new process orchestrated by LRRK2, which we name LYTL (LYsosomal Tubulation/sorting driven by LRRK2), by which lysosomal tubulation is used to release vesicles from lysosomes. Given the central role of the lysosome in PD, LYTL is likely to be disease relevant.
Lysosome-autophagosome fusion is critical to autophagosome maturation. Although several proteins that regulate this fusion process have been identified, the prefusion architecture and its regulation remain unclear. Herein, we show that upon stimulation, multiple lysosomes form clusters around individual autophagosomes, setting the stage for membrane fusion. The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein on lysosomes-vesicle-associated membrane protein 8 (VAMP8)-plays an important role in forming this prefusion state of lysosomal clusters. To study the potential role of phosphorylation on spontaneous fusion, we investigated the effect of phosphorylation of C-terminal residues of VAMP8. Using a phosphorylation mimic, we observed a decrease of fusion in an ensemble lipid mixing assay and an increase of unfused lysosomes associated with autophagosomes. These results suggest that phosphorylation not only reduces spontaneous fusion for minimizing autophagic flux under normal conditions, but also preassembles multiple lysosomes to increase the fusion probability for resuming autophagy upon stimulation. VAMP8 phosphorylation may thus play an important role in chemotherapy drug resistance by influencing autophagosome maturation.
Whereas the mechanisms involved in autophagosome formation have been extensively studied for the past 2 decades, those responsible for autophagosome-lysosome fusion have only recently begun to garner attention. In this study, we report that the multisubunit BORC complex, previously implicated in kinesin-dependent movement of lysosomes toward the cell periphery, is required for efficient autophagosome-lysosome fusion. Knockout (KO) of BORC subunits causes not only juxtanuclear clustering of lysosomes, but also increased levels of the autophagy protein LC3B-II and the receptor SQSTM1. Increases in LC3B-II occur without changes in basal MTORC1 activity and autophagy initiation. Instead, LC3B-II accumulation largely results from decreased lysosomal degradation. Further experiments show that BORC KO impairs both the encounter and fusion of autophagosomes with lysosomes. Reduced encounters result from an inability of lysosomes to move toward the peripheral cytoplasm, where many autophagosomes are formed. However, BORC KO also reduces the recruitment of the HOPS tethering complex to lysosomes and assembly of the STX17-VAMP8-SNAP29 trans-SNARE complex involved in autophagosome-lysosome fusion. Through these dual roles, BORC integrates the kinesin-dependent movement of lysosomes toward autophagosomes with HOPS-dependent autophagosome-lysosome fusion. These findings reveal a requirement for lysosome dispersal in autophagy that is independent of changes in MTORC1 signaling, and identify BORC as a novel regulator of autophagosome-lysosome fusion.
In specialized cells, such as mast cells, macrophages, T lymphocytes and Natural Killer cells in the immune system and for instance melanocytes in the skin, secretory lysosomes (SL) have evolved as bifunctional organelles that combine degradative and secretory properties. Mutations in lysosomal storage, transport or sorting molecules are associated with severe immunodeficiencies, autoimmunity and (partial) albinism. In order to analyze the function and content of secretory lysosomes in different cell populations, an efficient enrichment of these organelles is mandatory.
Microtubule post-translational modifications impart functional diversity to microtubules by affecting their dynamics, organization, and interaction with proteins. Using super-resolution microscopy, we show that only a small subpopulation of microtubules are detyrosinated in epithelial cells, while acetylated and tyrosinated microtubules comprise the majority of all microtubules. Surprisingly, lysosomes are enriched by approximately threefold on detyrosinated microtubules. Further, their motility on detyrosinated microtubules is impaired, showing shorter runs and more frequent and longer pauses. Lysosome enrichment is mediated through a kinesin-1-dependent mechanism, since knocking down this motor abolishes enrichment. Finally, correlative live-cell and super-resolution microscopy showed that lysosomes interact with autophagosomes on detyrosinated microtubules. Removal of detyrosinated microtubules or knockdown of kinesin-1 leads to a decrease in the percentage of autolysosomes, a fusion intermediate of autophagosomes and lysosomes. Taken together, our data reveal a new role of detyrosinated microtubules as hubs that spatially concentrate lysosomes on a small subset of microtubules and facilitate their interaction and fusion with autophagosomes to initiate autophagy.
Loss-of-function mutations in progranulin (GRN) are a major genetic cause of frontotemporal dementia (FTD), possibly due to loss of progranulin's neurotrophic and anti-inflammatory effects. Progranulin promotes neuronal growth and protects against excitotoxicity and other forms of injury. It is unclear if these neurotrophic effects are mediated through cellular signaling or through promotion of lysosomal function. Progranulin is a secreted proprotein that may activate neurotrophic signaling through cell-surface receptors. However, progranulin is efficiently trafficked to lysosomes and is necessary for maintaining lysosomal function. To determine which of these mechanisms mediates progranulin's protection against excitotoxicity, we generated lentiviral vectors expressing progranulin (PGRN) or lysosome-targeted progranulin (L-PGRN). L-PGRN was generated by fusing the LAMP-1 transmembrane and cytosolic domains to the C-terminus of progranulin. L-PGRN exhibited no detectable secretion, but was delivered to lysosomes and processed into granulins. PGRN and L-PGRN protected against NMDA excitotoxicity in rat primary cortical neurons, but L-PGRN had more consistent protective effects than PGRN. L-PGRN's protective effects were likely mediated through the autophagy-lysosomal pathway. In control neurons, an excitotoxic dose of NMDA stimulated autophagy, and inhibiting autophagy with 3-methyladenine reduced excitotoxic cell death. L-PGRN blunted the autophagic response to NMDA and occluded the protective effect of 3-methyladenine. This was not due to a general impairment of autophagy, as L-PGRN increased basal autophagy and did not alter autophagy after nutrient starvation. These data show that progranulin's protection against excitotoxicity does not require extracellular progranulin, but is mediated through lysosomes, providing a mechanistic link between progranulin's lysosomal and neurotrophic effects.
Expression of the deubiquitinase USP17 is induced by multiple stimuli, including cytokines (IL-4/6), chemokines (IL-8, SDF1), and growth factors (EGF), and several studies indicate it is required for cell proliferation and migration. However, the mechanisms via which USP17 impacts upon these cellular functions are unclear. Here, we demonstrate that USP17 depletion prevents peripheral lysosome positioning, as well as trafficking of lysosomes to the cell periphery in response to EGF stimulation. Overexpression of USP17 also increases secretion of the lysosomal protease cathepsin D. In addition, USP17 depletion impairs plasma membrane repair in cells treated with the pore-forming toxin streptolysin O, further indicating that USP17 is required for lysosome trafficking to the plasma membrane. Finally, we demonstrate that USP17 can deubiquitinate p62, and we propose that USP17 can facilitate peripheral lysosome trafficking by opposing the E3 ligase RNF26 to untether lysosomes from the ER and facilitate lysosome peripheral trafficking, lysosome protease secretion, and plasma membrane repair.
Genetic variation at the leucine-rich repeat kinase 2 (LRRK2) locus contributes to an enhanced risk of familial and sporadic Parkinson's disease. Previous data have demonstrated that recruitment to various membranes of the endolysosomal system results in LRRK2 activation. However, the mechanism(s) underlying LRRK2 activation at endolysosomal membranes and the cellular consequences of these events are still poorly understood. Here, we directed LRRK2 to lysosomes and early endosomes, triggering both LRRK2 autophosphorylation and phosphorylation of the direct LRRK2 substrates Rab10 and Rab12. However, when directed to the lysosomal membrane, pRab10 was restricted to perinuclear lysosomes, whereas pRab12 was visualized on both peripheral and perinuclear LRRK2+ lysosomes, suggesting that lysosomal positioning provides additional regulation of LRRK2-dependent Rab phosphorylation. Anterograde transport of lysosomes to the cell periphery by increasing the expression of ARL8B and SKIP or by knockdown of JIP4 blocked the recruitment and phosphorylation of Rab10 by LRRK2. The absence of pRab10 from the lysosomal membrane prevented the formation of a lysosomal tubulation and sorting process we previously named LYTL. Conversely, overexpression of RILP resulted in lysosomal clustering within the perinuclear area and increased LRRK2-dependent Rab10 recruitment and phosphorylation. The regulation of Rab10 phosphorylation in the perinuclear area depends on counteracting phosphatases, as the knockdown of phosphatase PPM1H significantly increased pRab10 signal and lysosomal tubulation in the perinuclear region. Our findings suggest that LRRK2 can be activated at multiple cellular membranes, including lysosomes, and that lysosomal positioning further provides the regulation of some Rab substrates likely via differential phosphatase activity or effector protein presence in nearby cellular compartments.
The nervous system spread of alpha-synuclein fibrils is thought to cause Parkinson's disease (PD) and other synucleinopathies; however, the mechanisms underlying internalization and cellular spread are enigmatic. Here, we use confocal and superresolution microscopy, subcellular fractionation, and electron microscopy (EM) of immunogold-labeled α-synuclein preformed fibrils (PFFs) to demonstrate that this form of the protein undergoes rapid internalization and is targeted directly to lysosomes in as little as 2 min. Uptake of PFFs is disrupted by macropinocytic inhibitors and circumvents classical endosomal pathways. Immunogold-labeled PFFs are seen at the highly curved inward edge of membrane ruffles, in newly formed macropinosomes, in multivesicular bodies and in lysosomes. While most fibrils remain in lysosomes, a portion is transferred to neighboring naive cells along with markers of exosomes. These data indicate that PFFs use a unique internalization mechanism as a component of cell-to-cell propagation.
An appreciation of the functional properties of the cytoplasmic fatty acid binding protein 4 (FABP4) has advanced with the recent demonstration that an extracellular form secreted by adipocytes regulates a wide range of physiological functions. Little, however, is known about the mechanisms that mediate the unconventional secretion of FABP4. Here, we demonstrate that FABP4 secretion is mediated by a membrane-bounded compartment, independent of the conventional endoplasmic reticulum-Golgi secretory pathway. We show that FABP4 secretion is also independent of GRASP proteins, autophagy, and multivesicular bodies but involves enclosure within endosomes and secretory lysosomes. We highlight the physiological significance of this pathway with the demonstration that an increase in plasma levels of FABP4 is inhibited by chloroquine treatment of mice. These findings chart the pathway of FABP4 secretion and provide a potential therapeutic means to control metabolic disorders associated with its dysregulated secretion.
Lysosomes are acidic and highly dynamic organelles that are essential for macromolecule degradation and many other cellular functions. However, little is known about lysosomal function during early embryogenesis. Here, we found that the number of lysosomes increased after fertilization. Lysosomes were abundant during mouse preimplantation development until the morula stage, but their numbers decreased slightly in blastocysts. Consistently, the protein expression level of mature cathepsins B and D was high from the one-cell to morula stages but low in the blastocyst stage. One-cell embryos injected with siRNAs targeted to both lysosome-associated membrane protein 1 and 2 (LAMP1 and LAMP2) were developmentally arrested at the two-cell stage. Pharmacological inhibition of lysosomes also caused developmental retardation, resulting in accumulation of lipofuscin. Our findings highlight the functional changes in lysosomes in mouse preimplantation embryos.
A more complete picture of the molecules that are critical for the organization of membrane compartments is beginning to emerge through the characterization of proteins in the vesicle-associated membrane protein (also called synaptobrevin) family of membrane trafficking proteins. To better understand the mechanisms of membrane trafficking within the endocytic pathway, we generated a series of monoclonal and polyclonal antibodies against the cytoplasmic domain of vesicle-associated membrane protein 7 (VAMP-7). The antibodies recognize a 25-kD membrane-associated protein in multiple tissues and cell lines. Immunohistochemical analysis reveals colocalization with a marker of late endosomes and lysosomes, lysosome-associated membrane protein 1 (LAMP-1), but not with other membrane markers, including p115 and transferrin receptor. Treatment with nocodozole or brefeldin A does not disrupt the colocalization of VAMP-7 and LAMP-1. Immunoelectron microscopy analysis shows that VAMP-7 is most concentrated in the trans-Golgi network region of the cell as well as late endosomes and transport vesicles that do not contain the mannose-6 phosphate receptor. In streptolysin- O-permeabilized cells, antibodies against VAMP-7 inhibit the breakdown of epidermal growth factor but not the recycling of transferrin. These data are consistent with a role for VAMP-7 in the vesicular transport of proteins from the early endosome to the lysosome.
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